Hemophilia A is a bleeding abnormality caused by a hereditary decrease or deficiency of blood coagulation factor VIII (FVIII) function. Hemophilia A patients are generally administered with an FVIII formulation for the bleeding (on-demand administration). In recent years, FVIII formulations are also administered prophylactically to prevent bleeding events (preventive administration; Non-patent Documents 1 and 2). The half-life of FVIII formulations in blood is approximately 12 to 16 hours. Therefore, for continuous prevention, FVIII formulations are administered to patients three times a week (Non-patent Documents 3 and 4). In on-demand administrations, FVIII formulations are also additionally administered when necessary at regular intervals to prevent rebleeding. In addition, the administration of FVIII formulations is done intravenously. Therefore, there has been a strong need for pharmaceutical agents with a lesser burden than FVIII formulations.
Occasionally, anti-FVIII antibodies (inhibitors) develop in hemophilia patients. Such inhibitors cancel the effects of the FVIII formulations. For bleeding in patients who have developed inhibitors (inhibitor patients), bypass formulations are administered. Their action mechanisms are not dependent on FVIII function, that is, the function of catalyzing the activation of blood coagulation factor X (FX) by activated blood coagulation factor IX (FIXa). Therefore, in some cases, bypass formulations cannot sufficiently stop the bleeding. Accordingly, there has been a strong need for pharmaceutical agents that are not affected by the presence of inhibitors and which can functionally substitute for FVIII.
As a means for solving these problems, bispecific antibodies that substitute for the function of FVIII and their use have been reported (Patent Documents 1, 2, 3, and 4). Bispecific antibodies against FIXa and FX can substitute for the function of FVIII by positioning the two factors close to each other to exhibit FVIII cofactor function-substituting activity (Non-patent Document 5). It has been reported that the FVIII cofactor function-substituting activity of the antibodies can be improved by optimizing the affinity and orientation towards FIXa and FX (Non-patent Document 6). Furthermore, the FVIII cofactor function-substituting activity of the antibodies is known to be affected by the IgG isotype, disulfide bond pattern, amino acid sequence of the hinge region, and the presence or absence of sugar chains in the Fc region (Non-patent Document 7). ACE910 (Emicizumab) having high FVIII cofactor function-substituting activity, which is one of these antibodies, has been reported to exhibit hemostatic effects in monkey models of hemophilia (Non-patent Documents 8 and 9). Furthermore, in clinical trials on healthy subjects, ACE910 (Emicizumab) was confirmed to achieve excellent pharmacokinetics (long half-life) and tolerability (Non-patent Document 10), and in clinical trials on hemophilia A patients with or without inhibitors, ACE910 (Emicizumab) administration remarkably reduced the bleeding rates compared to before ACE910 (Emicizumab) administration (Non-patent Document 11).
As described above, the effects of reducing the bleeding rates have been observed for ACE910 (Emicizumab) in clinical trials. However, in in vitro thrombin generation assays using FVIII-deficient plasma, improvement effects by ACE910 (Emicizumab) on the maximum amount of thrombin generation (peak height) was lower than the amount generated in the presence of a normal level of FVIII which is 100 U/dL (Non-patent Document 8). Therefore, further enhancement of drug efficacy has been desired. In addition, considering convenience for hemophilia A patients, there has been a demand for bispecific antibodies having FVIII cofactor function-substituting activity which can further reduce the administered dose through improvement of specific activity, and such.
Generally, there are cases where an antibody pharmaceutical acts as an antigen to induce anti-antibody (ADA) production (Non-patent Document 12). Since continuous administration of ACE910 (Emicizumab) becomes difficult for hemophilia patients with occurrence of ADA (anti-ACE910 (Emicizumab) idiotype antibodies), there has been a demand for bispecific antibodies having FVIII cofactor function-substituting activity which can be administered to such patients.
ACE910 (Emicizumab) is a bispecific antibody which has been optimized from many aspects by introducing many amino acid substitutions into a lead antibody hBS1. The lead antibody hBS1 was obtained by humanizing a bispecific antibody acquired through animal immunization which recognizes FIX and/or FIXa, and FX. ACE910 (Emicizumab) has high FVIII cofactor function-substituting activity (Non-patent Document 6 and Patent Document 4). However, for enhancement of drug efficacy and improvement of specific activity, a bispecific antibody that substitutes for the function of FVIII is necessary, which antibody has higher maximum activity (maximum FVIII cofactor function-substituting activity) than ACE910 (Emicizumab) and can exhibit FVIII cofactor function-substituting activity at concentrations lower than that of ACE910 (Emicizumab). However, to date, there have been no reports of bispecific antibodies having remarkably high FVIII cofactor function-substituting activity compared to ACE910 (Emicizumab) from the viewpoint of concentration and maximum activity (Patent Documents 4 and 5).
Several methods have previously been reported as methods for preparing IgG-type bispecific antibodies having human constant regions (IgG-type antibodies having a human constant region that has binding specificity for an antigen A on one arm and binding specificity for an antigen B on the other arm). In general, IgG-type bispecific antibodies are composed of two types of H chains (namely, an H chain for antigen A and an H chain for antigen B) and two types of L chains (namely, an L chain for antigen A and an L chain for antigen B). When such IgG-type bispecific antibodies are expressed, 10 types of combinations are possible as combinations of H2L2 since two types of H chains and two types of L chains are expressed. Among these, there is one type of combination that has the desired binding specificity (IgG having binding specificity for antigen A on one arm and binding specificity for antigen B on the other arm). Consequently, in order to acquire the desired bispecific antibody, it is necessary to purify one type of antibody of interest from among ten types of antibodies, which is extremely low in efficiency and difficult.
Methods have been reported for solving this problem, which involve preferentially secreting IgG having a heterologous combination of an H chain for antigen A and an H chain for antigen B, by substituting amino acids in the CH3 region of the IgG H chain (Patent Documents 6, 7, 8 and 9, and Non-patent Documents 13 and 14). Among these, there have been reported methods that use physical obstacles in the form of a “knob” and “hole”, and those that use electric charge repulsion.
A method has also been reported for efficiently obtaining a desired molecule, which uses a common L chain in which an L chain for antigen A and an L chain for antigen B are present on a same amino acid sequence (Patent Documents 10 and 11). However, since the use of a common L chain has the potential of considerably lowering the antigen affinity, this is not necessarily the optimum method. Consequently, in order for a bispecific antibody to bind to two antigens with high affinity, it is preferable that only the L chain and H chain for antigen A associate, and only the L chain and H chain for antigen B associate. Moreover, a method has been reported to allow the H chains and L chains for each antigen to associate irrespectively of the variable regions, which comprises substituting amino acids in the CH1 and CL domains which are constant regions, instead of those in the variable regions (Patent Documents 7, 12, and 13). However, this method leaves much to be improved for efficiently producing a bispecific antibody of interest.